Findings from FY11 are summarized below. 1. Orthopoxvirus pathogenesis The threat of intentional release of variola virus (VARV), the causative agent of smallpox, continues to be a chief biodefense concern. Zoonotic orthopoxviruses including cowpox (CPXV) and particularly monkeypox virus (MPXV) currently cause sporadic disease in humans, with case-fatality rates from MPXV infections reaching as high as 10%. Based on the above considerations, investigations into orthopoxviral pathogenesis and the identification of countermeasures have recently accelerated. The development of animal models that accurately reflect human disease is critical to our understanding of the pathogenesis of VARV infection and evaluation of countermeasures against the orthopoxviruses. Because of the successful eradication of smallpox and the sporadic and geographically isolated nature of MPXV outbreaks, the only option for licensing new drugs and vaccines for smallpox and other orthopoxvirus diseases is extrapolation of data derived from accurate, validated animal models. Furthermore, the underlying viral and host mechanisms that control disease outcome in orthopoxvirus infection are poorly understood and in need of continued study. While VARV and MPXV nonhuman primate (NHP) models in cynomolgus and rhesus macaques have been used for evaluation of vaccines and antiviral therapies, biosafety and security restrictions on the use of both viruses limit their widespread use by the research community. A NHP model that mimics VARV induced disease and is readily available to the broader research community (a BSL-2 agent) would serve as a distinct advantage to accelerate research and, if necessary, potentially replace the VARV NHP model. Therefore we have investigated whether cowpox, a BSL-2 agent which causes occasional NHP infections in European zoos, might be a useful model of orthopoxvirus infection. Unexpectedly, we found that intravenous inoculation of cynomolgus macaques with cowpox Brighton resulted in a lethal disease across all doses tested (5x10e5 to 5x10e7 PFU) within 12 days post-inoculation in two independent experiments. Clinical observations and hematological and histopathological findings supported hemorrhagic disease that resembled human hemorrhagic smallpox. Specifically, petechial rash, hemorrhagic lesions in skin and gut, hematuria, anemia, thrombocytopenia, elevated prothrombin times (PT), and increased concentrations of fibrin degradation products and D-dimers were consistently observed. Cowpox virus replicated to high levels in blood (8.0-9.0 log10 gene copies/mL) and tissues including lymph nodes, thymus, spleen, bone marrow, and lungs. While hemorrhagic smallpox was a rare manifestation of disease (<3% of cases), it was rapid, severe, and nearly uniformly fatal. The mechanisms that resulted in this form of disease are unknown. The characterization of this model and the hemorrhagic manifestations serves as an additional model to test countermeasures as well as a model to determine the pathogenesis of this most severe form of poxviral disease. Such studies will serve to complement the existing nonhuman primate models of more classical poxviral disease. We have also continued our studies of monkeypox pathogenesis. Specific highlights of FY2011 include expanded monitoring of disease progression in macaques by FDG-PET/CT by monitoring of bone marrow and lymph node immune activation in untreated or Cidofovir-treated macaques. In addition, we have identified cytokines that are associated with disease outcome after intravenous monkeypox infection. Finally, we have investigated the host kinome response to monkeypox virus infection with strains of differing virulence. 2. Pathogenesis of viral hemorrhagic fevers Viral hemorrhagic fevers are amongst the most deadly viral infections, but our understanding of viral pathogenesis, the host response to infection, and factors affecting disease outcome is limited. Simian Hemorrhagic Fever Virus (SHFV) has caused outbreaks of viral hemorrhagic fevers in Asian origin macaques that have occurred sporadically at primate research facilities after first being identified in 1964. SHFV is a BSL-2 pathogen that has not been linked to human disease;as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model resulting in a better understanding of human hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. We described the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64%. Infected NHPs developed disease suggestive of multi-organ system involvement including the mononuclear phagocyte, circulatory, lymphoid, renal, and hepatic systems. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Statistical analyses showed an association between lethal disease and increases in aPTT, AST, ALP, ALT, MCP-1, and IL-6 concentrations and a decrease in ALB. These results are important for several reasons. First they have demonstrated notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggesting that SHFV is an appropriate model of BSL-4 pathogens at the BSL-2 level. Second, the identification of biomarkers associated with disease outcome and severity points to factors that can be targeted for treatment or disease monitoring. Finally, examination of cytokine levels has implicated MCP-1 levels as predictive of severe disease in an additional viral infection suggesting further study and therapeutic potential. 3. Bivalent vaccines that confer protection against rabies and Ebola virus The search for a safe and efficacious vaccine for Ebola virus continues as no current vaccine candidate is nearing licensure. Ebola virus is a concern for humans and nonhuman primate populations living in Africa and a chief biodefense concern of the United States. We have developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Zaire ebolavirus (ZEBOV) glycoprotein (GP) using a reverse genetics system based on the SAD B19 RABV wildlife vaccine in collaboration with Matthias Schnell of Thomas Jefferson University. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein have greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. Furthermore, we have demonstrated that the bivalent vaccines are able to induce anti-EBOV immunity in the presence of pre-existing RABV antibodies, and that the RABV/EBOV vaccines can be added to multivalent formulations. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for endemic nonhuman primates at risk of EBOV infection. Problems with pre-existing vector immunity, safety, dosing schedule, and manufacturing obstacles that are associated with current competing Ebola vaccine candidates are largely absent from the RABV/EBOV approach. Rabies virus is still a severe and common disease in Africa, so vaccination of individuals with this bivalent vaccine would serve as a public health success